The disease yellow fever is controlled by the use of a live attenuated vaccine, strain 17D, derived by 176 passages of wild-type strain Asibi in chicken tissue. Next Generation Sequencing (NGS) technology has great applications to vaccine development and quality control and safety of live vaccines. We have compared low passage Asibi virus and 17D vaccine from a commercial dose of YF VaxTM by NGS and found that Asibi is a typical RNA virus with a quasispecies population while, surprisingly, commercial 17D vaccine has very little evidence of quasispecies, and this may contribute to the excellent safety record of the vaccine. This is the first comparison of a wild-type parent-live attenuated vaccine derivative for a human vaccine by NGS. We believe that investigation of the mechanism of limited quasi-species in 17D vaccine virus may have important applications to other live attenuated vaccines and development of future live attenuated vaccines. In this application we propose to obtain critical preliminary data to support a hypothesis for the mechanism of limited quasispecies in 17D vaccine by investigating what happens to the two viruses when passaged in different cell types or in vivo. We have preliminary data for unpassaged viruses and passage in three different cell types so Specific Aim 1 will investigate wild-type and vaccine strain virus genomic stability/diversity during infection of immunocompetent and immunocompromised mice to determine if the quasispecies populations of the two viruses identified above are retained or vary in different types of mice. In particular, lack of a functional innate immune response may enable 17D virus to generate a diverse population that results in a virulent phenotype. Specific Aim 2 will use reverse genetics to investigate whether the structural genes, nonstructural genes, or both are responsible for the limited quasispecies of 17D virus by utilizing infectious clones of Asibi and 17D viruses, and chimeric Asibi/17D viruses. It is hypothesized that the restricted quasispecies in 17D vaccine virus is due to mutation(s) in the nonstructural (NS) proteins of the replication complex as a whole, rather than the RNA dependent RNA polymerase alone, that results in high fidelity of the 17D RNA polymerase.